OBJECTIVE: The purpose of this study was to determine the effects of 2-h moderately prolonged exercise with carbohydrate intake or water placebo on salivary and urinary α-amylase isoenzyme activity in trained men. MATERIALS AND METHODS: Eleven aerobically fit men participated in this study. On two different occasions, participants performed 2-h cycling corresponding to a constant power output at 60% peak oxygen uptake. The study design involved a random order, placebo-controlled and cross-over assignment. Participants consumed either 6.2% carbohydrate solution or water placebo every twenty minutes thereafter (2 ml/kg body mass) over 2-h endurance exercise. Unstimulated whole salivary samples were collected using the passive drooling method at the 10-min period before and after exercise for the quantification of salivary α-amylase, immunoglobulin A (IgA) and total protein. Two-hour urinary samples were obtained at three time points before (-2-0h), immediately (0-2 h) after and 24-26 h after exercise for the analysis of α-amylase isoenzyme activity (pancreas- and saliva-derived types). RESULTS: The activity of α-amylase in saliva and urine was significantly increased in connect with salivary total protein concentration immediately after moderately long-lasting exercise, but salivary IgA concentration was not statistically significant with or without exogenous carbohydrate intake. CONCLUSION: These findings suggest that 2-h moderate exercise appears to lead to the enhanced α-amylase activity in saliva and urine regardless of exogenous carbohydrate availability, demonstrating enhanced mucosal immune defense.